Lab. No. 5
Gram’s Stain
Gram’s +ve
Cocci
Bacilli
Gram’s -ve
Cocci
Rods
Gram-negative, non-spore-forming bacilli.
 Their natural habitat is the intestinal tract
of humans and animals.
 Characters of enterobacteriaceae:
1- Facultative anaerobes that grow on
simple laboratory media.
2- Ferment glucose ± other
carbohydrates.
3-Oxidase negative.
4- Reduce nitrates to nitrites.

This family is classified on the basis of
biochemical reactions, especially
fermentation of carbohydrates.
 It can be divided according to their effect
on lactose into:

 Lactose
Fermenters: (coliforms)
e.g., E.coli, klebsiella.
 Lactose Nonfermenters:
e.g., protus, salmonella.
 Late Lactose Fermenters:
e.g., shigella.
Gram’s –ve bacilli ,
having single
arrangement.
Facultative anaerobes, grow on simple media



Proteus showing a swarming growth.
Klebsiella has a mucoid colonies.
E.Coli has a very bad odor.

Growth on MacConkey’s agar:
Principle:




MacConkey’s agar is a selective and
differential medium
selective medium for gram –ve bacteria
(bile salt & crystal violet inhibit the
growth of gram +ve bacteria).
Test sugar: lactose.
pH indicator: neutral red ( yellow in
alkaline, pink in acidic pH).
Lactose
Lactose fermenter
acid
Nutral red
Pink colonies
Procedure:
1. Inoculate MacConkey’s agar plate with
the test organism by streaking.
Flam & Cool
Flam & Cool
2. Incubate the plate at
Flam & Cool
35oC for 24
hrs.
Results:
Pink colonies
Lactose fermenter
Pale colonies
Lactose nonferment
1- Oxidase test.
2- Nitrate test.
3- O/F test.
1- Oxidase test:
Principle:
Tetramethyl p-phenylene diamine
(oxidase reagent)
colourless
Cytochrome oxidase enzyme
Indophenol
(Purple colour)
Results:
+ve Test: Appearance of purple colour within 1-2 min.
No colour
purple colour
-ve test
+ve test
Enterobacteriaceae
Pseudomonas
2- Nitrate test:
Principle:
All enterobacteriaceae reduce nitrates to
nitrites

Procedure:
Nit.A
test m.o
Incubate
at 35oC
for 24
hrs
Nitrate broth
Nit. B
Red colour
3- Oxidation Fermentation (O/F)
Test:
Principle:
 Using sensitive O/F medium ( Hugh and Leifson
Medium) .
 All enterobacteriaceae are O+/F+
(Fermentative)
Identification of lactose
fermenters:
1- TSI test.
2- IMViC
It is composed of a group of four tests:
 Indole production test.
 Methyl red (MR) test.
 Voges-Proskauer (V-P) test.
 Citrate Utilization test.
3- Growth on Eosin- Methylene blue agar.
4-urease production test.
1-Triple sugar iron (TSI) agar:
Principle:



This medium contains three types of sugars
(lactose, sucrose and dextrose).
The conc. of lactose and sucrose is 10 times
that of dextrose.
It also contains phenol red indicator.
Principle:
butt
slant
Procedure:
Results:
Lactose Fermentation:
Lactose
Large amount of acid
Butt: acidic (yellow)
Slant: acidic (yellow)
E.Coli & Klebsiella
2-IMViC Test:
It is composed of a group of four tests:
 Indole production test.
 Methyl red (MR) test.
 Voges-Proskauer (V-P) test.
 Citrate Utilization test.
1- Indole Production Test:
Principle:
inoculate the organism in peptone water for 24h.
Tryptophane
Tryptophanase
enzyme
Indole
Kovac’s reagent
Purple red color
in the upper
organic layer
Results:
+ve
-ve
2- MR-VP Test:
Principle:
inoculate the organism in glucose phosphate peptone for 48h,
Dextrose
Acidic pathway
Mixed acids
 pH less than 4.4
MR indicator
Red colour
MR +ve
OR
Neutral pathway
Acetyl methyl carbinol
α-naphthol
KOH
Brick red colour
VP +ve
3- Citrate Utilization Test
Principle:
Citrate
As a sole source of carbon
Na2CO3
alkaline
Bromothymol blue
Blue colour
indicator
+ve
Results:
IMViC

Indole
I

MR
MR

VP
VP

Citrate
C
Results:
+ve
+ve
-ve
-ve
-ve
-ve
+ve
I M Vi C
++ - -
I M Vi C
- - + +
E.Coli
Klebsiella
+ve
3- Growth on Eosin Methylene Blue (EMB)
Agar:
Principle:
 It is a selective and differential medium used
for isolation of fecal coliforms
 eosin and methylene blue are both:
 selective substances→ inhibit the growth of
most gram-positive organisms
 pH indicators→ combine to form a dark purple
precipitate at acidic pH.
 Test sugars: Sucrose& lactose.



Vigorous fermenters of lactose or sucrose
→ dark purple dye complex→ dark purple
to black colonies.
Non-lactose/sucrose fermenters →
normally-colored or colorless colonies.
E.coli, often produces a green metallic
sheen due to precipitation of M.B. in the
medium from the very high amounts of
acid produced.
 Klebsiella,
produces colonies with dark
center surrounded by light coloredmucoid rim (fish-eye appearance).
Results:
E.Coli
black colonies with metalic sheen.
Klebsiella
Purple colonies.
4-Urease production test:Principle:
Urea
Urease enzyme
NH3 ( alkaline)
Phenol red indicator
Pink colour
Results:
+ve
Klebsiella
-ve
E-Coli
Identification of lactosenonfermenters:
1- TSI test.
2- Growth on S-S agr.
3-Citrate utilization test.
4- Urease test.
5-Gelatin liquefaction test.
1-Triple Sugar Iron (TSI) agar:
Results:
1. Dextrose Fermentation:
a) Initial reaction:
(after 10 – 12 hrs)
Dextrose
acid
Butt: acidic (yellow)
Slant: acidic (yellow)
Dextrose Fermentation:
b) Delayed reaction:
(after 24 hrs)
Small amount of acid
Butt: acidic (yellow)
Slant: alkaline (red)
Proteus, Salmonells & Shigella
Principle:

This medium contains ferrous sulfate as an
indicator for H2S production.
H2S + FeSo4
FeS
Black ppt. of ferrous sulfid
Proteus & Salmonells
Results:
Butt:
acidic (yellow)
acidic (yellow)
acidic (yellow)
Slant:
acidic (yellow)
alkaline (red)
alkaline (red)
H2S Production:
-ve
E.Coli & Klebsiella
-ve
+ve
Shigella Proteus & Salmonells
2- Growth on Salmonella-Shigella
(S-S) Agar:
Principle:



It is selective and differential medium for
enteric pathogens especially Salmonella.
It contains lactose and neutral red
indicator
It also contains sodium thiosulfate and
ferric citrate as an indicator for H2S→
colonies with black centers.
Results:
Salmonella & proteus
Pale colonies with black centers
Shigella
Pale colonies
3-Citrate Utilization Test
Principle:
Citrate
As a sole source of carbon
Na2CO3
alkaline
Bromothymol blue
Blue colour
indicator
+ve

Results:-
Proteus &
Salmonella
+ve
Shigella
-ve
Definitive tests to differentiate between
Proteus & Salmonella
4- Urease Production Test:
Principle:
Urease enzyme
Urea
NH3 ( alkaline)
Phenol red indicator
Pink colour
Results:
+ve
Proteus
-ve
Salmonella
Definitive tests to differentiate between
Proteus & Salmonella
5- Gelatin Liquefaction Test:
Principle:
Gelatin
gel
Gelatinase
enzyme
Peptides
liquid
Results:
Positive test: Gelatin medium remain liquid even after refrigeration.
Gelatinase +ve
Gelatinase -ve
Proteus
Salmonella
Practicle Work











Oxidase test.
O/F test.
Nitrate broth.
Growth on MacConkey’s agar.
Indole broth.
MR-VP broth.
Citrate slant.
Growth on EMB agar.
Growth on S-S agar.
Urea Slant.
Gelatin test.
Flame & Cool
Flame & Cool
Flame & Cool
Results

Nitrate Test:
Nit.A
Red colour
Nit. B
No red colour
Add zinc dust
Results

Indole Production Test
Kovac’s reagent
I
Indole
Purple red color
in the upper
organic layer
Results

MR-VP Test:
MR indicator
MR
MR +ve
Red colour
MR-VP
broth
VP
α-naphthol
KOH
Brick red colour
VP +ve
Oxidase Test
-ve
+ve
Enterobacteriaceae
Pseudomonas
MacConkey’s agar
& TSI
 O/F test: O+/F-
Pink colonies on MacConkey Pale colonies on MacConkey
& acidic butt and slant onTSI & acidic butt alkaline slant onTSI
 Growth on cetrimide agar:
Lactose non-fermenter Pale colonies with green
pigmentation
Lactose fermenter
IMViC test
& EMB
IMViC
++ - & black colonies
with metalic
shines on EMB
E.coli
 Nitrate test: +ve further
reduction to N2
No H2S production
(no blacking in TSI)
IMViC
- - ++
Klebsiella
Shigella
H2S production
(blacking in TSI)
Urease production
& gelatin liquefaction
+ve
Proteus
-ve
Salmonella
Results
Pseudomonas E.coli
Gram’s
Stain
Oxidase
test
O/F test
Nitrate
test
Klebsiella shigella Proteus
Salmonella
Gram’s –ve rods, single arrangement
+ve
O+/FNo red colour
after addition
of zinc dust
+ve further
reduction to N2
Cetrimide Pale colonies
with green
agar
pigmentation
-ve
-ve
-ve
-ve
O+/F+ O+/F+ O+/F+ O+/F+
-ve
O+/F+
+ve reduction from nitrate into nitrite
-
-
-
-
-
Results
E.coli
Klebsiella
MacConkey’s Pink colonies Pink mucoid
agar
colonies
TSI agar
Butt:
Slant:
H2S:
IMViC test
EMB agar
shigella
Pale colonies
Proteus
Salmonella
Pale colonies Pale colonies
Acidic (yellow) Acidic (yellow) Acidic (yellow) Acidic (yellow) Acidic (yellow)
Acidic (yellow) Acidic (yellow) Alkaline (red) Alkaline (red) Alkaline (red)
-ve
-ve
+ve
+ve
-ve
IMViC
++ - -
IMViC
- - ++
Black colonies Pink colonies
with metalic
sheen
IMViC
-+--
IMViC
---+
Pale colonies Pale colonies
IMViC
-+-+
Pale colonies
Results
E.coli
Urease
production
test
Gelatin
liquefaction
test
S – S agar
-ve
-ve
Klebsiella
+ve
-ve
shigella
Proteus
Salmonella
-ve
+ve
(pink)
-ve
-ve
+ve
(liquid)
-ve
Pink colonies Pink colonies Pale colonies
Pale colonies
Pale colonies
with black
with black
centers
centers